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WO2002010345A2 - Matrice biologique autologue de foie mammifere et procede visant a son obtention - Google Patents

Matrice biologique autologue de foie mammifere et procede visant a son obtention Download PDF

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Publication number
WO2002010345A2
WO2002010345A2 PCT/EP2001/008646 EP0108646W WO0210345A2 WO 2002010345 A2 WO2002010345 A2 WO 2002010345A2 EP 0108646 W EP0108646 W EP 0108646W WO 0210345 A2 WO0210345 A2 WO 0210345A2
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WIPO (PCT)
Prior art keywords
bio
hepatocytes
autologous
matrix
liver
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PCT/EP2001/008646
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English (en)
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WO2002010345A3 (fr
Inventor
Giovanni Ambrosino
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Priority to AU2001289759A priority Critical patent/AU2001289759A1/en
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Publication of WO2002010345A3 publication Critical patent/WO2002010345A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C12N5/0671—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the present invention concerns an autologous bio-matrix derived from liver of porcine or human origin, the method for its obtainment and its use as a bio- artificial support of hepatocytes of homologous origin in the treatment of acute or chronic hepatic pathologies.
  • PRIOR ART It is known that the liver performs particularly important and sophisticated physiological functions of synthesis and detoxification and that the impairment of such functions account for the seriously disabling clinical evolution, when not fatal in the mid-short period from their onset, of both acute and chronic hepatic diseases of degenerative or metabolic nature.
  • liver in the functions of synthesis are immediately evident if it is considered that this organ is involved in the following metabolic processes: the synthesis and excretion of bilirubin and porphyrins, production and excretion of bile and biliary acids, excretion of urobilinogen, detoxification of endogenous, such as hormones, and exogenous substances (chemicals or drugs), energy production by mitochondria through biological processes of oxidation, the synthesis, use and elimination of amino acids in the metabolism, the synthesis of urea and catabolism of ammonia, the synthesis and secretion of proteins, the metabolism of carbohydrates, the synthesis and degradation of lipids, the storage of substances (e.g.
  • a bio-artificial liver indeed capable of functioning as a bridge for the functional support of the insufficient organ, must be able to perform both these functions; a third can be added to this, i.e. that of a stimulus for the diseased liver so that it regenerates, i.e. recovers, its full functionality, in a self-sufficient manner. Since such cellular activities are still not reproducible in the laboratory, the idea was put forward of using animal hepatic cells instead of the artificial biological filters, therefore capable of performing all the required complex functions. This then opened up the era of the bio-artificial liver, as a useful support, in cases in which the liver, affected by disease, quickly becomes impaired in its primary functions.
  • a bio-artificial liver is therefore an extracorporeal system, consisting of a similar apparatus to that of renal dialysis but which, unlike this, has a so-called "bioreactor" heart inside it.
  • the bioreactor the functional unit of the instrument, is generally made up of pig's liver cells fixed on a glass fibre support.
  • the hepatocytes once inserted in a bioreactor, expressly built and connected to an apparatus for extracorporeal use, and kept vital inside the system by sophisticated culture techniques, by coming into contact with the plasma of the patient affected by fulminating hepatitis, carry out their synthesis and detoxification action, on one hand temporarily replacing the impaired liver and, on the other, helping it to autonomously recover and to function under critical conditions.
  • hepatocyte transplantation is a method widely studied in experimental models to create an auxiliary liver if needed, both as hepatic support in fulminating hepatitides as well as potential treatment of metabolic deficiencies still of hepatic origin. Indeed, during the '30s Hans Popper carried out the first transplantations of liver fragments in the anterior chamber of the eye.
  • the answer may appear straightforward and immediate: use human hepatocytes obtained from livers unfit for transplantation, so as to avoid all the problems connected with the animal cell and, not least, its xenogenicity. All that however, in practice, does not prove so simple, since the human hepatocyte is a cell which, when isolated, is very sensitive to the damage induced by the current isolation methods, and even more to the effects of the cryoconservation and defrosting procedures, as well as quite quickly coming up against dedifferentiation.
  • a support which is capable of keeping the hepatocytes alive and functioning once implanted is not found on the market.
  • These spleen or portal vein grafts have shown positive activity in the first 20 days after grafting, but then quickly lose their functions or, even become detached from the implant site to then disperse in the blood system.
  • the applicant has now surprisingly found that a matrix consisting of an autologous bio-matrix derived from liver of porcine or human origin can be effectively used as a bio-artificial support of hepatocytes of autologous origin. Therefore the object of the present invention is the autologous bio-matrix originating from suitable mammalian livers, including human ones unfit for transplantation, usable as bio-artificial support of hepatocytes homologous with its origin and particularly of human or porcine origin.
  • Additional objects of the invention are the method for the preparation of the same and its use to support the hepatocytes of homologous origin in bioreactors for extracorporeal bio-artificial liver, and in the hepatocyte transplantations in the implantable biological liver both for the treatment of acute or chronic hepatic pathologies. Furthermore, the bio-matrix is able to be used for experimental purposes for in vitro studies on the function of the hepatocytes.
  • Figure 1a and 1 are photographs of the macroscopic aspect of the autologous bio-matrix.
  • Figure 2a and 2b are photographs of the microscopic aspect of the autologous bio-matrix.
  • Figure 3 is a photograph of the electronic aspect of the autologous bio-matrix with hepatocytes.
  • the autologous bio-matrix derived from mammalian liver and particularly of porcine or human origin, object of the present invention is instead able to advantageously constitute a support, biologically and above all functionally compatible with hepatocytes isolated in vitro of homologous origin, maintaining its structural configuration.
  • the bio-matrix allows the anchoring of the hepatocytes, taken from porcine or human liver and cultivated in vitro, according to a very precise polar arrangement and lets the same arrange themselves in culture in a functional and specific way.
  • the bio-matrix obtained with this method is in actual fact the "framework" indeed of the hepatic parenchyma, isolated using a low toxicity method and able to restore the physiological conditions indispensable to the hepatic cells.
  • the hepatocytes isolated in vitro and cultivated on the bio-matrix preserve their characteristics and functions for a long time, allowing the applications as already mentioned previously.
  • porcine origin Procine Liver Autologous Bio-Matrix - PLABM
  • human origin HLABM
  • object of the invention able to fix the hepatic cells according to polarity, the applicant has prepared an original enzymatic method and has shown that the hepatocytes thus supported can perform their functions in vitro for extended times also under critical conditions, both if that is when used in extracorporeal systems, such as the bio-artificial liver, and in totally implantable ones, such as the implantable biological liver.
  • the preparation method of the autologous bio-matrix is identical for both cases in which it is prepared from pig's liver as well as human liver.
  • the liver once withdrawn in toto, is placed in a freezer at -20°C.
  • the liver is brought to room temperature one hour before the bio-matrix preparation procedure and sections of 500-1000 micron thickness, cut manually or with a cold blade microtome, are prepared from the withdrawn liver.
  • the sections thus obtained, are treated for 2 hours in distilled water under agitation, changing the distilled water every 30 minutes.
  • the sections are then immersed in an aqueous solution at a concentration of 4% sodium desoxycholate and kept at rest for 2 hours.
  • the sections are treated with Type I Deoxyribonuclease (DNase) in salt solution (e.g. 2000 Kunitz Units of a solution with 1 M of Sodium Chloride) under agitation. This procedure can be repeated until the macroscopic and microscopic disappearance of cellular traces of hepatocytes.
  • DNase Type I Deoxyribonuclease
  • Figures 1 and 2 The autologous bio-matrix obtained with the method described is depicted in Figures 1 and 2. From the point of view of its use it should again be mentioned that the bio-matrix must not necessarily be prepared at the moment of its use, but that this bio-matrix can be, once prepared, preserved both in its pure or lyophilized form under appropriate conditions of temperature and sterility. The lyophilized form simply requires rehydration with physiological solutions.
  • the bio-matrix keeps its structural characteristics and biological properties essential for a functional support to hepatocytes completely intact.
  • Similar matrix preparation methods have already been described for the preparation of matrices from other organs, namely bladder and stomach, but not in the case of liver presumably due to this organ's functional and structural peculiarities.
  • certain technical devices are necessary which the applicant has found as a result of its research in the field, and particularly such devices are necessary to obtain a bio-matrix with the characteristics fit for the purpose, i.e. elasticity and absence of traces of hepatocytes.
  • hepatocyte elimination must take place mostly under agitation in distilled water and not in DNase, to avoid toxic effects of the latter (on the contrary treatment with DNase is instead always required for other organs).
  • object of the invention to function as a support of the hepatocytes isolated according to the technique described, both when used in bioreactors for extracorporeal bio-artificial liver, and when used in the transplantations of hepatocytes in the implantable biological liver for the treatment of acute or chronic hepatic pathologies, tests were carried out, hereafter reported, using isolated hepatocytes supported on the bio-matrix in experimental models of acute hepatic failure.
  • Induction of acute hepatic failure through the administration of exogenous substances such as galactosamine and carbon tetrachloride, although much used, are not particularly suitable in the case in which pigs are used as the experimental animal owing to uncontrollable variables, such as the dose-weight ratio, pretreatment of animals and the metabolism capacity of the drug typical of each animal.
  • the surgical model was chosen on account of these reasons of experimental nature, despite it proving more complex from a technical point of view and having the disadvantage of being unreversable.
  • the surgical model is in fact always perfectly reproducible and not tied to any factor (weight, type of anaesthesia, age, etc.). Animal mortality always occurs at the same time period, around the 18 th hour in pigs, but this technique allows the study of hepatocyte synthesis activity both in the extracorporeal systems as well as in the transplantation of the same.
  • Experimental methods Female pigs weighing 30 Kg, without food from the previous day but with water ad libitum were used. Surgery was carried out under general anaesthesia by means of oro-tracheal intubation. Surgical technique Stage One: Median incision.
  • the biological activity of the hepatocytes isolated as previously described supported on the autologous bio-matrix was tested using the serum of pigs kept anhepatic for 12 hours.
  • the cellular vitality and functionality were measured by: laboratory tests (AST, ALT; Urea, Albumin, PT, Glycemia, Bilirubin); Glycogenic load; MTT.
  • laboratory tests AST, ALT; Urea, Albumin, PT, Glycemia, Bilirubin
  • Glycogenic load MTT.
  • the pig serum kept anhepatic for 12 hours showed a reduction in all the values studied, except that of bilirubin which instead increased.
  • TAB. 1 the glycogenic load of the hepatocytes was high and MTT staining was positive.
  • Transaminase 700 300 500 500 600
  • pigs 10 of which treated and 10 control animals, were used for the experiment. All the pigs were made anhepatic, as described, and connected through the femoral vein and artery to an extracorporeal apparatus thermostatically set at 37°C, consisting of an apparatus for plasmaseparation, an oxygenator and a peristaltic pump, with appended a bioreactor.
  • the bioreactor used was a module consisting of a chamber for cellular cultures, containing, only in the case of treated animals, some plates with hepatocytes sown on autologous bio-matrix. From the plasmapheresis apparatus, the oxygenated plasma was propelled by the peristaltic pump towards the bioreactor.
  • the plasma of the anhepatic treated animal came into contact with the hepatocytes in the bioreactor through a fixed porosity membrane.
  • the plasma thus treated was reinfused into the animal after recovery with its corpuscular part by the same apparatus for plasmapheresis. The treatment was extended for 12 hours.
  • the parameters measured were: AST, ALT, Glycemia, PT, MEGx (Lidocaine) at to, at 3, 6, 9, 12 hours of treatment.
  • the hepatocytes and the autologous bio-matrix were controlled by optical and electronic microscopy ( Figure 3) at the end of treatment. In addition, MTT and the glycogenic load were assessed.
  • the control group, in which the pig was kept anahepatic for 12 hours without treatment with hepatocyted supported on autologous bio-matrix showed: a significant drop in glycemia, an absence of metabolization of lidocaine, a progressive decrease in transaminase until its disappearance, a significant reduction in PT (Tab. 2).
  • transaminase was of little significance.
  • the in vitro results obtained show that the hepatocytes sown on autologous bio-matrix are able to perform biosynthetic activities up to 30 days of culture with just 20% mortality.
  • This important biological property makes the bio-matrix particularly useful in the support of hepatic cells, also human, used in extracorporeal type apparatuses, such as the bio-artificial liver, or in the transplantation of hepatic cells in the implantable biological liver.
  • the bio-matrix can be used for experimental purposes for in vitro studies on the function of the hepatocytes, a function at present still largely unknown in its basic mechanisms.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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Abstract

L'invention concerne une matrice biologique autologue dérivée d'un foie d'origine porcine ou humaine, capable de constituer un support fonctionnel bio-artificiel de cellules hépatiques homologues à leur origine. La matrice biologique permet l'ancrage d'hépatocytes, extraits d'un foie porcin ou humain et cultivés in vitro, conformément à un agencement polaire très précis, et laisse ceux-ci s'agencer en culture d'une façon fonctionnelle et spécifique. Cette propriété biologique importante rend la matrice biologique particulièrement utile dans le support de cellules hépatiques, même humaines, utilisé dans des appareils de type extra-corporel, tels qu'un foie bio-artificiel, ou dans la greffe de cellules hépatiques, les deux pouvant servir dans le traitement de pathologies hépatiques aiguës ou chroniques. La matrice biologique peut également être utilisée à des fins expérimentales pour des études in-vitro sur la fonction des hépatocytes.
PCT/EP2001/008646 2000-07-28 2001-07-26 Matrice biologique autologue de foie mammifere et procede visant a son obtention Ceased WO2002010345A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001289759A AU2001289759A1 (en) 2000-07-28 2001-07-26 Autologous bio-matrix from mammalian liver

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITMI2000A001744 2000-07-28
IT2000MI001744A IT1318655B1 (it) 2000-07-28 2000-07-28 Bio-matrice autologa da fegato di mammiferi e metodo per l'ottenimentodella stessa.

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WO2002010345A2 true WO2002010345A2 (fr) 2002-02-07
WO2002010345A3 WO2002010345A3 (fr) 2002-08-15

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998025637A1 (fr) * 1996-12-10 1998-06-18 Purdue Research Foundation Materiel biologique derive du tissu hepatique de vertebres

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AU2001289759A1 (en) 2002-02-13
ITMI20001744A1 (it) 2002-01-28
WO2002010345A3 (fr) 2002-08-15
IT1318655B1 (it) 2003-08-27
ITMI20001744A0 (it) 2000-07-28

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